Non-invasive tissue glucose level monitoring

ABSTRACT

Instruments and methods for performing non-invasive measurements of analyte concentrations and for monitoring, analyzing and regulating tissue status, such as tissue glucose levels.

RREFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a continuation-in-part of applicationno. 09/287,486, filed Apr. 6, 1999, which claims the benefit under 35U.S.C. § 119 (e) of provisional application no. 60/080,794, filed Apr.6, 1998, which are both entitled Non-Invasive Tissue Glucose LevelMonitoring and herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to instruments and methods for performingnon-invasive measurements of analyte concentrations and for monitoring,analyzing and regulating tissue status, such as tissue glucose levels.

BACKGROUND OF THE INVENTION

[0003] Diabetes is a chronic life threatening disease for which there ispresently no cure. It is the fourth leading cause of death by disease inthe U.S. and at least 90 million people worldwide are estimated to bediabetic. Diabetes is a disease in which the body does not properlyproduce or respond to insulin. The high glucose levels that can resultfrom this affliction can cause severe damage to vital organs, such asthe heart, eyes and kidneys.

[0004] Type I diabetes juvenile diabetes or insulin-dependent diabetesmellitus) is the most severe form of the disease comprisingapproximately 10% of the diabetes cases in the United States. Type Idiabetics must receive daily injections of insulin in order to sustainlife. Type II diabetes, (adult onset diabetes or non-insulin dependentdiabetes mellitus) comprises the other 90% of the diabetes cases. TypeII diabetes is often manageable with dietary modifications and physicalexercise, but may still require treatment with insulin or othermedications. Because the management of glucose to near normal levels canprevent the onset and the progression of complications of diabetes,persons afflicted with either form of the disease are instructed tomonitor their blood glucose level in order to assure that theappropriate level is achieved and maintained.

[0005] Traditional methods of monitoring the blood glucose level of anindividual require that blood be withdrawn. This method is painful,inconvenient, costly and poses the risk of infection. Another glucosemeasuring method involves urine analysis, which, aside from beinginconvenient, may not reflect the current status of the patient's bloodglucose because glucose appears in the urine only after a significantperiod of elevated levels of blood glucose. An additional inconvenienceof these traditional methods is that they require testing supplies suchas collection receptacles, syringes, glucose measuring devices and testkits. Although disposable supplies have been developed, they are costlyand can require special methods for disposal.

[0006] Many attempts have been made to develop a painless, non-invasiveexternal device to monitor glucose levels. Various approaches haveincluded electrochemical and spectroscopic technologies, such asnear-infrared spectroscopy and Raman Spectroscopy. Despite extensiveefforts, however, none of these methods appears to have yielded anon-invasive device or method for the in vivo measurement of glucosethat is sufficiently accurate, reliable, convenient and cost-effectivefor routine use.

SUMMARY OF THE INVENTION

[0007] The invention overcomes problems and disadvantages associatedwith current strategies and designs and provides new instruments andmethods for monitoring, analyzing and regulating in vivo glucose levelsor other analyte levels in an individual.

[0008] In one general aspect, the invention features a non-invasiveglucose monitoring instrument useful in vivo. The instrument maycomprise a radiation source capable of directing radiation to a portionof the exterior or interior surface of a patient. That surface may be amucosal area such as the gums and other mucosal areas, the eyeballs andsurrounding areas such as the eyelids and, preferably, the skin. Thesource emits radiation at a wavelength that excites a target within thepatient such that the excited target provides a glucose level indicationof the patient. A glucose level indication is a quantitative or relativemeasurement that correlates with the blood glucose content orconcentration of the patient. The instrument may further comprise aradiation detector positioned to receive radiation emitted from theexcited target, and a processing circuit operatively connected to theradiation detector that translates emitted radiation to a measurablesignal to obtain the glucose level indication. The target is not glucoseitself, but a molecular component of the patient such as, for example, acomponent of skin or other tissue, that reflects or is sensitive toglucose concentration, such as tryptophan or collagen cross-links.Suitable targets are structural components, and compounds and moleculesthat reflect alterations in the environment of matrix components of thetissue and are sensitive to or correlate with tissue glucoseconcentration. The target provides an emitted fluorescence signal thatis related to the patient's blood glucose level. The radiation detectoris responsive to the emission band of the target or species in the skin.Preferably the radiation is ultraviolet radiation or light. The emittedradiation is preferably fluorescence radiation from the excitation ofthe non-glucose target. The instrument may further include means formeasuring scattering re-emitted from the irradiated skin. The radiationemitted from the excited target and signal therefrom correlates with theblood glucose of the patient.

[0009] Another aspect of the invention relates to an instrument forassessing changes in the superficial structural matrix of the skin orother tissue of a patient comprising means for measuring fluorescence,and means for measuring scattering.

[0010] Another aspect of the invention relates to an instrument forassessing changes in the environment of matrix components of the skin orother tissue of a patient comprising means for measuring fluorescence,and means for measuring scattering. Preferred embodiments furtherinclude means for combining signals from the means for measuringfluorescence and the means for measuring scattering.

[0011] Another aspect of the invention relates to a non-invasive methodof detecting or assessing a glucose level comprising exciting a targetthat, in an excited state, is indicative of the glucose level of apatient, detecting the amount of radiation emitted by the target, anddetermining the glucose level of the patient from the amount ofradiation detected. The target is preferably a molecular species in theskin. Preferred targets are tryptophan or a matrix target, like PDCCL,which are excited by ultraviolet radiation and act as bioamplifiers orbioreporters. Targets may be structural matrix or cellular components.Suitable targets reflect alterations within the environment of matrixcomponents of the skin or other tissue and act as bioamplifiers orbioreporters when excited with ultraviolet radiation.

[0012] Still another aspect of the invention relates to a non-invasivemethod of assessing a change in the superficial structural matrix of atissue, or a change in the environment of matrix components, comprisingexposing the tissue to radiation at a first wavelength, detecting anamount of fluorescence emitted by exposed tissue, exposing the tissue toradiation of a second wavelength, detecting an amount of scatteringre-emitted from the exposed tissue, and deriving an indicationrepresentative of the change in the superficial structural matrix of thetissue, or a change in tissue matrix components or their environment,based on of the amount of fluorescence detected and the amount ofscattering detected.

[0013] Other objects and advantages of the invention are set forth inpart in the description which follows, and in part, will be obvious fromthis description, or may be learned from the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 A multipurpose skin spectrometer that provides dataspecifically relevant to signals correlating with blood glucose.

[0015]FIG. 2 Block diagram of one embodiment of a glucose levelmonitoring instrument.

[0016]FIG. 3 Graph of the average fluorescence excitation spectra fornormal and diabetic SKH mice for an emission wavelength of 380 nm.

[0017]FIG. 4 Graph of the average fluorescence excitation spectra fornormal and diabetic SKH mice for an emission wavelength of 340 nm.

[0018]FIG. 5 Graph of the average fluorescence excitation spectra for arat at an emission wavelength of 380 taken at different blood glucoselevels.

[0019]FIG. 6 Plot of the fluorescence intensity at 346 nm for fourdifferent glucose levels which are taken from FIG. 5.

[0020]FIG. 7 Graph of the average fluorescence excitation spectra for anemission wavelength of 380 nm for a human male before and after theingestion of 100 grams of glucose.

[0021]FIG. 8 Graph of the average fluorescence excitation spectra for anemission wavelength of 380 nm for a human male before and after theingestion of 100 grams of glucose.

[0022]FIG. 9 Graph of the average fluorescence excitation spectra for anemission wavelength of 380 nm for a human female before and after theingestion of 100 grams of glucose.

[0023]FIG. 10A A diagram depicting collection of fluorescence spectrawith components attributable to tryptophan and collagen cross linksfollowing irradiation with UV light.

[0024]FIG. 10B A diagram depicting scattering according to a scatteringmodel.

[0025]FIG. 11 Block diagram of a monitoring instrument that can be usedto monitor tissue glucose levels or evaluate changes in the superficialstructural matrix of a tissue or the environment of matrix components ofa tissue.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0026] As embodied and broadly described herein, the present inventionrelates to devices and methods for quantitating, trending and/orreporting an analyte, such as blood glucose, to devices and methods formonitoring and regulating in vivo glucose levels, and to devices andmethods for evaluating the superficial structural matrix or cellularcomponents of a tissue.

[0027] It has been discovered that by measuring fluorescence followingirradiation of a tissue surface of a patient, such as the patient'sskin, and by optionally assessing scattering, the glucose level of apatient can be evaluated. Evaluation according to the invention is basedon the surprising discovery that the quantum efficiency of fluorescenceof a responsive target within the skin is transiently affected by theirradiation and can be correlated to the ambient glucose content.Long-term interaction between diabetes, collagen and other species hasbeen previously observed (V. M. Monnier et al., Diabetes 37:867-872,1988). However, a reversible component of this interaction thatcorrelates with blood glucose levels and possibly depends on the glucoselevel in the environment of collagen and other targets has previouslygone unnoticed. More specifically, although glucose itself does notfluoresce to any significant degree, when the blood glucose level of apatient changes, the quantum efficiency of fluorescence of a target suchas, for example, pepsin-digestible collagen cross links (PDCCL), alsochanges. This change may be due, in part, to the direct and indirecteffects of the relative presence of glucose or other molecules on theenvironment of target molecules and structures. That presence induces areversible change in the quantum efficiency of fluorescence productionby the target which can be detected and analyzed. Glucose molecules inthe environment may be covalently or noncovalently coupled to the target(glycosylated collagen), or simply free in the immediate vicinity of thetarget. Targets may be in the dermal matrix, in the epidermal matrix, orin cells or the immediate vicinity of cells associated with the eitherthe dermis or the epidermis. In this regard, the invention may also beused to directly assess the amount or degree of advanced glycation endproducts that exist in an area of the body such as, for example, invessels, arteries or organs.

[0028] A fluorescent signal originating from dermal collagen cross linkshas been identified, which signal slowly increases with aging and isalso sensitive to transient exposure to ultraviolet radiation. PDCCLfluoresces following excitation at 335-340 nm, with the emission maximumat 390 nm (N. Kollias et al., Journal of Investigative Dermatology,111:776-811998). The fluorescent signal decreases monotonically with asingle UV exposure, but recovers within hours. With multiple exposures,the effects appear cumulative, and recovery takes weeks. However, it hasbeen discovered that transient changes in the environment of thesecollagen cross links causes significant and transient alterations intheir fluorescence which can be tightly correlated with blood glucosedeterminations.

[0029] Targets in the environment of matrix components, such as collagencross links serve as bioamplifiers or bioreporters of ambient glucoseconcentrations and, thus, constitute a novel and sensitive means ofnon-invasively assessing glucose in real time. Advantages of thismethodology include a large change in signal level for a relativelysmall change in collagen structure or matrix environment. The method isalso unhampered by absorption from competing species in the generalarea. In addition, there are only a few fluorophores which makes signalanalysis easier. Further, detector sensitivity is generally excellentand instrumentation and optical components, all of which arecommercially available, are potentially simpler and less expensive thanthose used for infrared measurements. Also, given the robust signals andsignal to noise ratios observed, there is potentially less of a need toresort to complex algorithmic and chemometric analyses.

[0030] Accordingly, one aspect of the present invention is related to anon-invasive in vivo glucose monitoring instrument that determinesglucose levels or changes in glucose levels by measuring fluorescence ofthe skin following excitation of one of these targets or species.Specifically, fluorescence signals obtained following irradiation ofskin or other tissue can be correlated with glucose levels, or changesin glucose levels, by measuring fluorescence following excitation oftargets or species within the environment of the matrix components.Preferred targets are structural matrix components such as PDCCL.Another preferred target is epidermal tryptophan which, like othertargets, may be bound to other compounds or structures, andintracellularly or extracellularly localized. Other useful matrixtargets for excitation include collagenase-digestible cross links,elastin cross links, glycosaminoglycans, glycated collagen andglycosylated substances in a tissue. These targets may also be referredto as biosensors as they are biological substances that detectablychange in response to glucose content, or bioamplifiers as they mayamplify a signal indicative of systemic glucose levels.

[0031] A non-invasive glucose monitoring instrument according to oneaspect of the invention includes a radiation source capable of directingradiation to a portion of the surface of the skin (or other tissue) of apatient. The source emits radiation at a wavelength that excites atarget of species in the tissue that can be correlated with bloodglucose content, such that the excited target provides a glucose levelindication of the patient. In a preferred embodiment, the target is amolecule other than glucose, and most preferably is a structural matrixcomponent such as, for example, collagen cross-links. Alternatively, thetarget may be tryptophan. When the target being detected is cross-linkedcollagen, the ultraviolet radiation source is preferably operative toirradiate at approximately 330-345 nanometers, and the ultravioletdetector is sensitive to emitted wavelengths in the range of 370-410nanometers, more preferably, 380-400 nanometers and, most preferably,390 nanometers. As noted, another useful target whose change in emissionmay be detectable is tryptophan. When the target being detected istryptophan, the ultraviolet radiation source is preferably operative toirradiate at approximately 285-305 nanometers, more preferably atapproximately 295 nanometers, and the ultraviolet detector is preferablysensitive to emitted wavelengths in the range of 315-420 nanometers,more preferably 340-360 nanometers, and most preferably, 345 nanometers.The radiation emitted by the target correlates with the glucose level ofthe patient. The spectral information can be converted into a numbercorrelative to standard blood glucose determinations.

[0032] The instrument further comprises a radiation detector positionedto receive radiation emitted from an excited target. The instrumentfurther includes a processing circuit operatively connected to theradiation detector and operative to translate a level of emittedradiation into a measurable signal that is representative of or may becorrelated with the blood glucose level. Preferably, the radiationsource is ultraviolet light. In a preferred embodiment the radiationsource may comprise a flexible-fiber optic arm or probe that directssaid radiation to the target. The probe may comprise a glass or quartzfiber and may be flexible and easily manipulated to examine a siteanywhere on the patient's skin. The portion of skin irradiated may beless than about 1 square cm, and more preferably is about 0.2 square cm.Preferably, the portion is a site which is most easily measurable on thepatient such as on the arm or leg. Differences in pigmentation betweendifferent areas of the body as well as different patients can befactored or eliminated through selection of control input, and overcome.

[0033] The instrument may further comprise a display such as, forexample, a visual, auditory or sensory display operatively connected tothe processing circuit and operative to display the glucose levelindication. Optionally, this data may be analyzed and transmitted to apump or other servo mechanism responsive to the processing circuit. Thepump is incorporated into the system such that the pump administersinsulin or other medication to the patient at a rate that corresponds tothe glucose level signal.

[0034] Referring to FIG. 1, an embodiment of the glucose monitor of theinvention includes a Xenon arc (Xe-arc) lamp, double excitation andemission monochromators, a photomultiplier device, a simple currentamplifier and a flexible probe. The probe may comprise fiber opticbundles which allow convenient evaluation of living systems. Thisembodiment can take the form of a multipurpose skin spectrometer or itmay be modified to create a unit optimized to provide data specificallyrelevant to signals correlating with blood glucose. One advantage ofutilizing fluorescent excitation spectra compared to fluorescenceemission spectra is that the former are similar to absorption spectra,which aids in the separation and identification of the individualfluorophores in a complex spectrum. Although other components can besubstituted for the elements in this embodiment, a Xe-arc in combinationwith an excitation monochromator, avoids the major constraint of lasersources, namely the limited number of excitation wavelengths.

[0035] Optionally, other types of sources, such as a diode laser,coupled with enhanced spectral analysis algorithms optimized for thecollagen cross links may be used. These algorithms may also incorporatevariables such as skin type, age, exposure, etc., all of which areanalyzed during testing. Hardware modifications and calibrations may beincorporated to take into account these and other variables. Specificalgorithms and software may be embedded into a dedicated processor. Forexample, one design may comprise a night hypo/hyperglycemia monitoringinstrument which is programmed to alarm by trending analysis parametersthat correlate with significant changes in blood glucose. Alternatively,monitoring could be performed with a transportable fiber-basedfluorescence spectrophotometer with double monochromators, both on theexcitation and emission paths. This allows the evaluation of differentsubsets of collagen cross links and tryptophan signals as well asallowing the estimation of epidermal melanin pigmentation or othertissue pigments. Optimized instruments may duplicate and incorporate thefunctionality and data processing requirements incorporated fromappropriate studies.

[0036] Another embodiment uses a fiber-based fluorescence spectrometerwith two double monochromators and a high intensity excitation lightsource (350 W Xe-arc). The double monochromator design minimizes straylight, which tends to be high because of the high level of lightscattering by the tissues. The probe is preferably a fiber optic devicethat allows collection of data from different skin sites on the body.Probe design is optimized to permit ease of use and reproducibility.Optimization of light sources, filters and software can be designed toperform three scans that maximize the collagen fluorescence signals. Onescan is preferably 250-360 nm on the excitation band and 380 nm on theemission. The second scan is preferably 250-400 nm on the excitation and420 nm on the emission. The third scan is preferably 360-480 nm on theexcitation and 500 nm on the emission. This provides information onPDCCL (340/390 nm), the collagenase digestible collagen crosslinks(370/460 nm) and the collagen/elastin crosslinks (420/500 nm), amongother species. The system may also provide data on tryptophan, anepidermal fluorophore having an excitation wavelength of 290-300 nm andan emission wavelength of 340-360 nm, among other species. Devices maybe small and lightweight desktop units useful in health care providersettings. A remote probe may be connected to the system through aflexible fiber optic bundle. Data output may consist of a reporting of aquantitative number that correlates with blood glucose readings, alongwith spectral data, which may be displayed on a separate small I/Oterminal or laptop computer. The software further contains diagnosticoverlay capabilities.

[0037] Another device allows monitoring of glucose levels, by providingspectral information reflective of glucose levels, on a continuous orrepetitive basis. In one embodiment, this would be used throughout thenight with a built-in alarm, to alert the patient to abnormal decreasesor increases in glucose levels. The unit, which may be the size of aclock radio, can have a fiber optic cable to connect to the patient,similar to existing apnea monitors and pulse oxymeters. Another portabledevice may be placed in contact with the skin for periodic momentaryglucose readings. It may have an LCD readout for glucose levels, memoryto store several hundred glucose readings and a data output to downloadstored data.

[0038] An alarm may be operationally coupled to the processing circuitsuch that the alarm is activated when the glucose level indicationexceeds a first predetermined value (such as 200 gm/l), falls below asecond predetermined value (such as 70 gm/ml), or varies more than 20%from a third predetermined value (such as the previously measured levelor a baseline level determined for the patient). Alternately, the alarmmay be triggered in response to a more complex algorithmic analysis ofdata or based on evaluation by trending analysis over time.

[0039] The instrument may further comprise a normalizing detectorresponsive to another target in the tissue, such that the processingcircuit is responsive to the normalizing detector to normalize theglucose level indication. For example, a current or latest glucose levelsignal may be normalized by comparing it to a previously determinedglucose level signal which has been calibrated by comparing it directlywith a conventionally determined blood glucose level. Alternatively,normalization may involve comparison of emissions from the same targetbut at another wavelength, comparison of emissions from a non-targetsuch as glucose or another structural or circulating component of thebody, or simply taking a reading from another skin site. Normalizationmay also be performed by comparison to similar data from another pointor points in time taken from the same individual, or utilizing a storeddatabase or spectral library. Normalizing may alternately compriseobtaining a baseline signal before any prolonged activity wherecontinual measurements would be difficult such as, for example, beforedriving or sleeping, and watching for changes or trends of changes. Thepreviously determined glucose level signal may also be compared with alevel assessed from a simultaneously drawn blood sample. In addition,scattering evaluation may be factored into the normalizing process.

[0040] The instrument may optionally comprise means for measuringscattering re-emitted from the tissue. As discussed below, the means formeasuring scattering may comprise a skin illuminating means that emitsradiation at an angle greater than 60 degrees to said target or it maycomprise a skin or tissue illuminating means which emits radiation atbetween about 330 to 420 nm. Re-emitted radiation is then collected andanalyzed.

[0041] The instrument may include a portable housing in which theradiation source, the radiation detector and the processing circuit aredisposed. The instrument may include a battery compartment disposed inthe housing and a pair of battery contacts operatively connected to theultraviolet radiation source, the ultraviolet radiation detector, andthe processor. Data can be electronically recorded, stored or downloadedfor later review or analysis. The instrument may further compriseattachment means for attaching the radiation source, a portion of, orall of the device to the patient. The portable housing, the ultravioletradiation source, the ultraviolet radiation detector, and the processormay be designed so that they weigh in combination less than 3 kilograms,more preferably less than 1 kilogram, and most preferably, less than 0.5kilograms. The instrument may optionally include an attachment mechanismfor attaching the housing to the patient. Alternately, the instrumentcan be miniaturized; in such an embodiment, a microprocessor isincorporated onto a dermal patch, which may be operatively connected toother devices that provide input directly to a pump or other biodeliverysystem, such as a transmucosal or inhalational system, which may deliverinsulin or other appropriate medication to the patient.

[0042] The instrument may also be constructed using small componentscomposed of inexpensive, possibly recyclable materials such as plasticsand paper, so that the entire instrument or a significant portion isdisposable. For example, the entire device can be incorporated into apatch worn anywhere on the body and secured with adhesive tape,hook-and-loop fastener or another suitable means. After expiration ordepletion of an integral battery, the patch can be safely and easilydisposed of and a new patch secured. Such instruments weigh less than 1kg, preferably less than 0.5 kg and more preferably less than 0.1 kg.

[0043] The processing circuit is preferably operative to translate thelevel of detected radiation into a measurable glucose level signal thatis indicative of the glucose level in the tissue. The signal may bedirectly evaluated, or it may be compared to stored reference profiles,to provide an indication of changes from previous levels or trends inthe patient's glucose level. Although a preferred embodiment measuresradiation or fluorescence following irradiation of the skin, the presentinvention can also be used to assess glucose levels by evaluation ofother tissues. For instance, glucose levels may be assessed inaccordance with the present invention by detecting radiation orfluorescence following irradiation of the surface of other tissues, suchas mucous membranes, or irradiation of the mucosa, submucosa or serosaof any organ.

[0044] Another aspect of the invention relates to a non-invasive methodof detecting a glucose concentration or level in vivo comprising thesteps of exciting a target in the skin or other tissue, preferably usingultraviolet radiation, that is sensitive to the patient's tissue glucosecontent and is indicative of the glucose level of the patient, detectingan amount of radiation or fluorescence emitted by the target, andderiving an indication representative of or which correlates with acurrent blood glucose level based on the amount of radiation orfluorescence detected. Preferably, the target is a matrix target such ascollagen cross links. Alternatively, the target may be tryptophan. Themethod may optionally include the step of determining whether to takesteps to adjust the patient's glucose level in response to the derivedglucose level, followed by the step of administering insulin or anotherpharmaceutical composition in response thereto. The method may includeany one or more of the steps of reporting the information to thepatient, recommending a dosage, or administering the composition, suchas insulin, to the patient in response to the indication derived. Thestep of administering may be performed by using a syringe, a pump oranother suitable biodelivery system, mechanical or chemical, which maybe implanted or external to the body. The method may also include thestep of displaying a glucose level indication related to the indicationderived or providing a warning to the patient. The method may furtherinclude the step of normalizing the glucose level indication derived inthe step of deriving. The steps of exciting, detecting, and deriving maybe performed continuously or at any appropriate interval, for example,by the minute, hourly, daily or every other day for the same patient andover a period of days, weeks, months or years.

[0045] Optionally, the method may include actuating an alarm in responseto the glucose level when the glucose level exceeds a predeterminedfirst level, falls below a predetermined second level or varies morethan a set percentage, such as for example, 10%, 20%, 30%, 50% or 100%or more from a predetermined third level or changes in such a way thatmeets criteria of a specifically designed algorithm. The method mayfurther comprise the step of measuring scattering re-emitted from theskin or irradiated tissue surface and utilizing the resulting data toinitiate or assist in actuating a process aimed at adjusting the glucoselevel.

[0046] Instruments according to the invention are advantageous in thatthey provide information relative to blood glucose and permit glucoselevels to be evaluated noninvasively. Such non-invasive instrumentsallow people with diabetes to monitor glucose levels without the pain,inconvenience, and risk of infection associated with obtaining a bloodsample. By making monitoring safer and more convenient, people withdiabetes can monitor their glucose levels more frequently and thereforecontrol levels more closely. Safer and more convenient glucose levelmonitoring reduces the likelihood of measurements being skipped.

[0047] Furthermore, by coupling the instrument according to theinvention with a pump or other device which can deliver insulin or othertherapeutic agent to the patient, using a transmitter, or other suitablecommunication device, such that the pump or device is responsive to theglucose signal, even finer automatic glucose level monitoring may beachievable. For example, the transmitter may remotely transmit thesignal to a pump, such as a servo pump, having a receiver responsive tothe transmitted signal. The pump is preferably responsive to informationderived from or analysis of the spectral signal. The pump may thenprovide insulin or other appropriate medication to the patient.Alternately, or in addition, the information may be sent to a remotemonitor.

[0048] As will be clear to those of skill in the art, the instrumentsand methods of the present invention can also be used in forensicapplications, to allow the non-invasive and non-destructive assessmentof forensic tissues. In addition, the instruments and methods may beused to detect and diagnose diabetes, monitor the progression ofdiabetes, and detect and monitor other disorders involving hyper orhypoglycemia or abnormal blood sugar metabolism. Although the term invivo is used to refer to living material, it is intended herein toencompass forensic applications as well.

[0049] Another embodiment of the present invention is depicted in FIG.2, which depicts a glucose level monitoring instrument 10 includingsource driving circuit 12 having an output provided to an input 13 of asource 14. Source driving circuit 12 controls the illumination, providedby source 14. Source driving circuit 12 may take a number of forms,depending on the nature of the source and the acquisition. Examplesinclude a regulated power supply or a pulsed modulator.

[0050] Source 14 preferably comprises an ultraviolet light source, suchas a continuous mercury lamp, a pulsed or continuous xenon flash lamp,or a suitable laser. Useful lasers include, but are not limited to,nitrogen lasers, OPO (tunable laser) and Nd YAG pump devices. The outputof source 14 may be filtered to restrict illumination to withinexcitation bands of interest. Its intensity (and pulse width ifapplicable) is preferably set at a level that minimizes exposure whileoptimizing signal-to-noise considerations. It is also possible toirradiate the sample with two or more short (e.g. femptosecond) pulsesof multiphoton light having a wavelength two or more times longer thanthe wavelength of interest, such that the radiation penetrates to adifferent degree or depth. The source is positioned to illuminate anarea of interest on the patient's skin 16.

[0051] Glucose level monitoring instrument 10 also includes a detector18 that is sensitive to ultraviolet light emitted by the species that isexcited by the source 14. The detector has an output 15 operativelyconnected to an input of an acquisition interface 20, which may be ananalog-to-digital converter with an analog input operatively connectedto the detector output. A digital output port 21 of the acquisitioninterface is operatively connected to processor 22.

[0052] Processor 22 is operative to convert the digital detector outputsignal into a glucose level signal. The processor may perform thisconversion by applying various signal processing operations to thesignal, by comparing signal data with stored reference profiles, or byother appropriate methods. It has an output 23 provided to a display 24,permitting the glucose level indication to be presented to the user. Theoutput may be directly provided to display 24, or sent remotely via atransmitter. Display 24 may be an alphanumeric display which displaysthe glucose concentration as a percentage.

[0053] The glucose level monitor instrument 10 may also include amedication delivery device, such as insulin pump 26, which is responsiveto the glucose level signal or other spectroscopic data or analysisprovided by processor 22. A transmitter may be used to transmit theglucose level signal of processor 22 to the pump. The pump is configuredso that it converts the glucose level signal received from processor 22into an insulin dispensing rate. A single bolus of insulin may also beadministered based on the glucose level signal. The use of an insulinpump allows the glucose level to be controlled both continuously andautomatically. The medication delivery device can also deliver anothertherapeutic substance, or administer an electrical, chemical, ormechanical stimulus. Miniaturized devices may be constructed ofdisposable materials such as plastics and paper to further reduce cost.Instrument 10 may be implemented in a number of different ways. It maybe implemented at a board level, with the various elements describedbeing separate components, mounted on a circuit board. Many of theelements may also be integrated into a dedicated special-purposeintegrated circuit allowing a more compact and inexpensiveimplementation. Alternatively, the components may be miniaturizedfurther to create an implantable device or a dermal patch. Inintegrating and miniaturizing the various functions of the instrument,many of them may be combined. Important algorithms may be embedded.

[0054] Instrument 10 may also include a normalizing section. Thenormalizing section is designed to reduce or eliminate the effect ofvariations, such as the intensity of source 14 or day to day variationsin the patient's tissue. A normalizing section may include a seconddetector that is responsive to a species in the skin that fluoresces butdoes not respond to glucose concentration. It may also normalize to asignal collected at another time, another site, or another wavelength orfrom a different internal or external target. Processor 22 may receivesignals from the two detectors and derive a normalized glucose levelsignal. Preferably instrument 10 includes a portable housing bearingultraviolet radiation source 14, ultraviolet radiation detector 18,acquisition interface 20 and processor 22. Instrument 10 may be poweredvia battery contacts by a battery contained in the battery compartmentlocated within the housing. Preferably, the entire assembly weighs incombination less than 20 kg, preferably less than 10 kg and morepreferably less than 1 kg. Highly portable embodiments which weigh underone kg may be attached to the patient in a monitoring position, such asby an elastic or hook-and-loop fastener strap.

[0055] In operation, a physician or the patient places source 14 closeto an area of interest on the patient's skin 16. Preferably, this areais one that is not regularly exposed to sunlight, such as the inside ofthe upper arm. The physician or patient may then start the instrument'smonitoring sequence. The monitoring sequence begins with driving circuit12 producing a driving signal that causes source 14 to irradiate thearea of interest on the surface of the skin 16 with one or more bands ofultraviolet radiation. The spectral content of this radiation isselected to cause one or more targets within the skin to fluoresce.These targets may include tryptophan, collagen cross-links or othersuitable targets. The excitation/emission wave lengths for tryptophanand collagen cross-links are 295/340-360 nanometers and 335-340/380-400nanometers, respectively. To increase the sensitivity of themeasurement, it is also possible to pre-expose the area of interest witha higher intensity of radiation, before making measurements. Note alsothat the excitation and emission wavelengths are representative of themolecular species targeted. Under circumstances where the target isresponsive to multiple different wavelengths and provides differentinformation from each, or where targets and non-targets are responsiveto the same wavelength, more accurate and qualitative values may beobtained by identifying and eliminating background and other interferingdata.

[0056] The target absorbs the radiation from the source and re-emits itback to detector 18. Detector 18 derives a signal representative of thereceived emitted radiation and provides it to the acquisition interface20. Acquisition interface 20 translates the derived signal into adigital value, which it provides to processor 22. Processor 22 convertsthe digital value into a display signal, which it provides to display24. The display signal may take the form of an alphanumericrepresentation which correlates with the concentration of glucose in theblood, or it may include another kind of display signal to be used withanother type of display. For example, it is possible to use a colorcoding scheme to indicate levels of glucose, or indicate dosage amountsto the patient on the display based on the signal received at thedetector. The display need not be a visual display; tactile actuators,speakers, or other machine-human interfaces may be employed. The glucoselevel signal produced by the processor may be directly displayed toindicate the patient's glucose level. Alternately, the processor mayfirst compare the data from the detector with stored reference profiles,such as the patient's prior levels, to provide information regardingtrends in the patient's glucose level.

[0057] Still another aspect of the invention is related to a glucosemonitoring system with alarm features. Parents of children with diabetesare under a continuous threat that a severe hyper- or hypoglycemic eventmay occur without their knowledge, such as during the night, withpotentially fatal consequences. There are an increasing number ofindividuals with diabetes in need of a device for monitoring theirglucose levels. Accordingly, this aspect of the invention is related toa monitoring device with an alarm that alerts a parent or otherinterested person in the event of large or dangerous changes or trendsin the blood glucose levels of a patient. The device reports systemichyperglycemic and/or hypoglycemic events using fluorescent detection ofalterations in the environment of matrix components that reflect changesin blood glucose. Alternately, the device may detect the change influorescence from the excitation of another suitable species, such astryptophan. The device may be completely portable, miniaturized and/ordisposable allowing its use in nearly any environment.

[0058] The alarm may be any suitable alarm including, but not limitedto, a sound alarm or a radio transmitter that activates a sound or lightemitting unit in the proximity of the parents or other interestedperson. The alarm may be audible, visible, vibrating or any othersensory detectible type. For example, in one embodiment, the patient'sglucose level is measured once or at a plurality of intervals shortlybefore the patient goes to sleep to determine a baseline glucose level.The device is programmed to take measurements of the patient's glucoselevel at periodic intervals during the night, and to then compare theseresults with the baseline. If the glucose level varies more than apredetermined percentage from the baseline either simply or utilizingspecifically designed algorithms, an alarm sounds. Although any desiredpercentage variation can be selected, in a preferred embodiment, thealarm is activated when the glucose level varies more than 5%, 10%, 20%or more from the previously determined baseline or in accordance with apreviously defined set of parameters or specifically designedalgorithms. Alternately, or in addition, the alarm is triggered if thepatient's blood glucose level exceeds a first predetermined level (i.e.it exceeds 200 gm/ml) or if it falls below a second predetermined level(i.e. it falls below 70 gm/ml). When the alarm sounds, the patient canthen be administered insulin (or other suitable medication) if theglucose level is too high, or can be given a source of sugar if theglucose level is too low. Alternatively, or in addition, the alarm maybe triggered if other analysis or trending patterns occur.

[0059] Optionally, the processor of this device, or any of themonitoring devices disclosed herein, may include means for storing anddisplaying a plurality of sequential measurements, so that trends whichoccur during the night or during other time periods of interest may besaved and evaluated. The measurements can be taken continuously orrepetitively at predetermined intervals. For example, a patient can beperiodically monitored after the administration of one or more of thevarious forms or sources of insulin (i.e. lente, ultralente, semilente,regular, humalog, NPH etc.) or other glucose regulatory therapies todetermine or help to determine the most suitable treatment protocol forthe patient. This may be influenced by a comparison to other readingsover time, a broader data base, a derivation based on the slope of thechange of the signal over time and where on the scale of patient risk aparticular assigned glucose might fall.

[0060] As mentioned above, the fluorescence signals measured from theexcitation of PDCCL and other tissue components are affected by thechanges in the scattering properties of the superficial structuralmatrix. As the electrolyte balance in the micro environment of collagencross links changes, changes are induced in fluorescence. In addition,the change in electrolytes also produces a change in the local index ofrefraction and thus a change in the scattering properties. The change inscattering causes a change in the fluorescence.

[0061] A diagram depicting fluorescence of species sensitive to glucoseconcentration following irradiation of the skin is depicted in FIG. 10A.Incoming radiation at wavelength λi is directed towards the skin. Itpenetrates the stratum comeum. If λi is 295 nanometers, fluorescentradiation (λo) will be emitted at 345 nanometers by tryptophan in theepidermis of the skin. If λi is 335 nm, fluorescent radiation will beemitted (λo) at 390 nm by the collagen cross links in the dermis.

[0062] A diagram depicting a scattering according to a scattering modelis depicted in FIG. 10B which shows collagen cross links in thesuperficial dermis bending incoming light (λi) in different directions.The re-emitted light (λo) is at the same wavelength as the incominglight (λi), but is scattered due to changes in the local index ofrefraction. By independently measuring scattering in the superficialmatrix, the monitoring of blood glucose levels by measuringfluorescence, as described above, can be enhanced. Specifically, theresults from the assessment of scattering can be used to correct forchanges in fluorescence induced by changes in the scattering propertiesof the relevant layers of the dermal matrix.

[0063] Accordingly, another aspect of the invention is related to adevice that measures the scattering properties of a target such assuperficial collagen dermal matrix in the skin, which is affected bychanges in the chemical environment which can be correlated with bloodglucose levels. Although it has been previously been reported that thescattering properties of the skin (dermal matrix) change with glucoseconcentration and that these changes are measurable with photonmigration techniques in the near infrared (NIR), the use of NIRwavelengths provides a sample of the whole dermis and subcutis (does notmeasure one signal specific for glucose, but rather many signals thatare neither specific for glucose nor reliably linked to glucose levelsin a linear fashion). In contrast, the present invention assesses thescattering properties of the superficial dermis, as opposed to thedeeper layers. Such scattering of polarized light by the superficialdermal matrix is most noticeable in the range 380-700 nm.

[0064] Assessment of scattering in tissue, such as the superficialdermis, associated with changes in blood glucose can most preferably bemeasured by using short wavelengths (330-420 nm) or launching theilluminating light at large angles (preferably >60°). Short wavelengthsare preferably used because they penetrate to a small depth into thedermis. Alternately, changes in scattering induced by the presence ofglucose may be measured using light in the visible range of 620-700 nmand looking for changes in signal intensity.

[0065] One of the benefits of assessing scattering of the superficialdermis, as opposed to deeper layers of the dermis, is that fluorescentsignals from PDCCL's and other matrix components originate there and areaffected by the changes in the scattering properties. Further, thesuperficial layers of the dermis (in areas of the body receiving minimalenvironmental insults) are well organized and this would be reflected inscattering of polarized light. Since glucose has a strong polarizationrotation property, such changes may be measurable when monitoring at asubmillimeter resolution, but when monitoring on a gross scale theeffects of local organization would be canceled out. Increases influorescence may be compensated for by decreases in the effectivescattering, making the fluorescence signal difficult to separate frombackground noise. By independent measurement of the scattering withrandomly polarized and with linearly polarized light, fluorescencedetection may be enhanced, allowing it to stand on its own merit as amethod of indirect measurement of glucose concentration.

[0066]FIG. 11 depicts an embodiment in which both fluorescence of thesuperficial dermis and scattering are evaluated in order to assessglucose levels. Although this embodiment is described in connection withmonitoring blood glucose, as will be clear to those of skill in the art,it can be adapted to assess the status of other analytes, or to detectchanges in the superficial structural matrix or matrix components of atissue. Instrument 100 comprises a power supply 101 connected viaconnection 102 to a light source 104. Light source 104 may be a lamp, anarc lamp, a laser, or other suitable illumination device. Power supply101 receives feedback 103 from data acquisition controller 122 toregulate the intensity, synchronization or pulse rate of the lightemitted from light source 104. Light source monitor output 105, whichmay comprise a PIN diode, Avalanche diode, photomultiplier, CCD or othersuitable device, couples the light source 104 to data acquisitioncontroller 122. Light 106 is directed to wavelength selection device107, where an appropriate wavelength is selected, and selected lightwavelength output 109 is directed via a fiber, prism or a combination ordirectly through the air, to illuminate skin 110. Wavelength selectiondevice 107 may comprise a monochromator, a filter or a combination ofboth. If a laser source is used as light source 104, a filter or otherwavelength selection device may not be needed. Wavelength selectiondevice 107 is coupled via signal connection 108 to data acquisitioncontroller 122 to enable selection of the wavelength and to verify thepresent wavelength.

[0067] Fluorescent signals are emitted and scattered light is re-emittedfrom skin 110. The fluorescent light and reflective intensity 111 ispicked up by wavelength selective device 112, which may comprise amonochromator, filter or a combination. Wavelength selective device 112provides a light output 114 to detector 115. Detector 115 may comprise aphotomultiplier, diode, avalanche diode, CCD or other suitable detectiondevice. The signal from detector 115 is transmitted to signalconditioner/processor 120 via signal connector 116. Detector 115 issupplied power via power cable connection 1 17 from power supply 1 18.Data acquisition controller 122 provides input to power supply 118 viasignal connection 119 to allow selection of sensitivity orsynchronization with the light source. Wavelength selection device 112is coupled via connection 113 to data acquisition controller 122 toselect wavelength and verify current wavelength. Signalprocessor/conditioner 120 provides output via output connection 121 todata acquisition controller 122. Data acquisition controller 122 isconnected via connection 123 to display 125. Data acquisition controller122 may also provide output via connection 124 to an insulin ormedication delivery device.

[0068] The above described instrument may also be used as a non-invasivedevice for assessing changes in the superficial structural matrix or theenvironment of matrix components due to a variety of disease conditions.This embodiment allows the assessment of changes in the structuralmatrix non-invasively by measuring the combination of fluorescence andscattering, and comparing these results to measurements of developedstandards, temporal correlates or surrounding normal tissue. This devicemay be used to assess changes in the collagen matrix brought about bydiseases such as diabetes, scleroderma, scarring, or atrophy induced bythe use of steroids. It is also useful to assess changes in the matrixdue to aging or photoaging and changes induced by long exposures to zerogravity environment. This embodiment may be miniaturized, and may beused clinically and in research applications to evaluate wound healing,protein metabolism, diabetes, collagen diseases and other conditions.

[0069] The collagen cross links in the superficial or papillary dermisprovide large fluorescence signals that are indicative of the state ofthe collagen matrix. These signals may be monitored non-invasivelywithout interference with the functions of the skin. Specifically, asthe matrix is irradiated with UVA, UVB or UVC radiation, thefluorescence of PDCCL decreases. This fluorescent effect recoversfollowing a single exposure; however, the changes induced becomepermanent after multiple exposures.

[0070] The fluorescence of the skin in the UVA (320-400 nm) resultsmainly from collagen cross links that lie in the papillary dermis. Thefluorescence signals from these cross links may be used to evaluate thestate of the collagen matrix. In the skin and other tissues, as thecollagen matrix is degraded due to the expression of matrixmetalloproteinases, such as collagenase, in the stroma of tumors so doesthe fluorescence emission from the collagenase digestible collagen crosslinks. By assessing fluorescence, it has been discovered thatdegenerative changes in the superficial structural matrix or of matrixcomponents may be assessed, such as changes induced by disease orenvironmental factors such as diabetes, age, photodamage, topicalsteroid application, or prolonged exposure to zero-gravity. Further, theintensity of scattered light by the dermis changes with aging and withchanges in the collagen cross links. If the collagen cross links in thesuperficial or papillary dermis change, then the amount of light that isscattered by the dermis and its dependence on wavelength will alsochange. These changes may be monitored by reflectance.

[0071] Another aspect of the invention is related to a device that canmeasure either fluorescence excited at about 335 nm (pepsin digestiblecollagen cross links), fluorescence excited at about 370 nm (collagenasedigestible collagen cross links), or both, as well as the reflectancespectrum (450-800 nm), to thereby provide information on the state (orchanges induced) of the superficial structural matrix or environment oftissue matrix components. By combining the assessment of fluorescenceand scattering into one instrument, a novel device is provided thatprovides enhanced information on the state of the structural matrix orenvironment of tissue matrix components. Other wavelengths can also beused for excitation, such as 295 nm for tryptophan. A preferredembodiment incorporates a light source (Hg) and filters to select either333 nm 365 nm or visible broad band. The visible excitation may beprovided by a tungsten halogen lamp of 1-2 watts. The light is thenconducted to the skin's surface by fibers, reflective optics ordirectly, and the fluorescence from the UVA excitations and thereflectance from the visible source are assessed with a photodiode arraytype of detectors. The fluorescence intensities can then be compared tostandard signals from collagen samples (prepared from gelatin). Thereflectance signal is analyzed for scattering and absorption byiterative methods at wavelengths of 620-820 nm. Accordingly, anotheraspect of the invention is related to an instrument for assessingchanges in a superficial structural matrix of the skin or theenvironment of the matrix components of a patient comprising means formeasuring fluorescence, and means for measuring scattering.

[0072] Another aspect of the invention is related to a non-invasivemethod of assessing a change in the superficial structural matrix of atissue or a change in the environment of the matrix components of atissue comprising exposing the tissue to radiation at a firstwavelength, detecting an amount of fluorescence emitted by exposedtissue, exposing the tissue to radiation of a second wavelength,detecting an amount of scattering re-emitted from the exposed tissue,and deriving an indication representative of the change in thesuperficial structural matrix or environment of the matrix components ofthe tissue based on of the amount of fluorescence detected and theamount of scattering detected. Preferably, the first wavelength isultraviolet radiation, or is between about 320 and 420 nm and the secondwavelength is between about 330 and 420 nm. Preferably, the tissue isskin.

[0073] The following examples are offered to illustrate embodiments ofthe present invention, but should not be viewed as limiting the scope ofthe invention.

Examples

[0074] Example 1 Glucose Levels of Diabetic versus Non-Diabetic Mice

[0075] Experiments were conducted for six shaved hairless (SKH) diabeticmice made diabetic by the injection of streptozotocin, and six shavedhairless (SKH) non-diabetic (normal) mice. Excitation spectra atemission wavelengths of 380 nm and 340 nm were collected for each of thetwelve mice. A Xenon arc source coupled to a monochromator were fed intoa fiber optic probe, which was then used to illuminate the backs of allof the mice at an intensity level of approximately 0.1-1.0 mw/cm. Aspectrometer was used to collect the resulting spectra, which are shownin FIGS. 3 and 4 for emission at 380 nm and 340 nm, respectively. Theplots indicate a significantly lower excitation intensity at 295 nm anda significantly higher excitation intensity at 340 nm for the diabeticmice. Urine collected from the animals confirmed that the glucose levelsof the diabetic mice were higher at 340 nm for the diabetic mice.

[0076] Example 2 Glucose Levels of a Non-Diabetic Rat Following Ketamineand Insulin Treatments

[0077] Referring to FIG. 5, experiments were also conducted using anormal rat. The experimental apparatus used was the same as that used inExample 1. Fluorescence excitation spectra were obtained for the rat inthe following situations, (A) at rest (diamonds), (B) after theadministration of Ketamine (squares), (C) after the administration ofinsulin (triangles) and (D) after the administration of additionalinsulin (crosses). The glucose levels in situations A-D were determinedto be 120, 240, 100, and 40 gm/ml, respectively. The results arebelieved to be superimposed on a light leakage signal that increasessteadily with wavelength, although the use of double monochromatorsshould eliminate this source of background noise. Spectra collected forthis rat indicate that blood glucose level has a positive effect onfluorescence excitation in the 340 nm range. This is more clearlydepicted in FIG. 6 in which the fluorescence excitation intensity at 346nm for each of the situations A-D has been plotted.

[0078] Example 3 Glucose Levels of Human Subjects Before and AfterGlucose Ingestion

[0079] Preliminary experiments were also conducted on humans. FIGS. 7, 8and 9 depict fluorescence excitation spectra for three human subjects,two males and one female, respectively, before (dashes), 30 minutesafter (dotted/dashed line), and 60 minutes after (solid line) theingestion of 100 grams of glucose. In each situation, the emissionmonochromator was set to a wavelength of 380 nm. Collagen and tryptophanspectra were found to change in ways similar to those for the animalmodels, although there appear to be individual differences. Dashed linesrepresent measurements before glucose intake. Dashed and dotted linesrepresent changes induced after glucose intake. Solid lines representmaximal changes induced by the intake of glucose.

[0080] Other embodiments and uses of the invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the invention disclosed herein. As will be clear to those ofskill in the art, the devices and methods of the present invention canbe easily adapted to reflect or detect the level of a variety ofsubstances in tissue, in addition to glucose and the described targets.All references cited herein, including all U.S. and foreign patents andpatent applications, including, but not limited to, U.S. ProvisionalPatent Application Serial No. 60/080,794, entitled Non-Invasive TissueGlucose Level Monitoring, filed Apr. 6, 1998, are specifically andentirely incorporated by reference. The specification and examplesshould be considered exemplary only with the true scope and spirit ofthe invention indicated by the following claims.

We claim:
 1. A nocturnal glucose monitor, comprising: a glucosemeasurement probe, an alarm unit responsive to the glucose measurementprobe, the alarm unit being operative to detect excursions of glucosevalues outside of a predetermined nocturnal glucose range for a diabeticpatient and to produce an alarm signal in response to detectedexcursions, and an audio transducer responsive to the alarm signal. 2.The nocturnal glucose monitor of claim 1 further including a remotetransmitter responsive to the glucose measurement probe and a receiverresponsive to the remote transmitter, wherein the audio transducer isresponsive to the remote transmitter.
 3. The nocturnal glucose monitorof claim 2 wherein the remote transmitter is a wireless transmitter andthe receiver is a wireless receiver.